Keith Warriner

Chapter 4 Recent Advances in the Microbial Safety of Fresh Fruits and Vegetables

Foodborne illness outbreaks linked to fresh produce are becoming more frequent and widespread. High impact outbreaks, such as that associated with spinach contaminated with Escherichia coli O157:H7, resulted in almost 200 cases of foodborne illness across North America and >

Internalization of bioluminescent Escherichia coli and Salmonella Montevideo in growing bean sprouts

300 m market losses. Over the last decade there has been intensive research into gaining an understanding on the interactions of human pathogens with plants and how microbiological safety of fresh produce can be improved. The following review will provide an update on the food safety issues linked to fresh produce. An overview of recent foodborne illness outbreaks linked to fresh produce. The types of human pathogens encountered will be described and how they can be transferred from their normal animal or human host to fresh produce. The interaction of human pathogens with growing plants will be discussed, in addition to novel intervention methods to enhance the microbiological safety of fresh produce.

Persistence and growth of different Salmonella serovars on pre- and postharvest tomatoes.

Aims: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts.

Inactivation of human pathogens and spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide

The interaction of a range of Salmonella serovars with pre- and postharvest tomatoes was evaluated. Serovars were selected on the basis of previous association in tomato-linked outbreaks of salmonellosis (Salmonella Javiana, Salmonella Montevideo, and Salmonella Newport) or those typically isolated from animal or clinical infections (Salmonella Dublin, Salmonella Enteritidis, Salmonella Hadar, Salmonella Infantis, Salmonella Typhimurium, and Salmonella Senftenberg). Salmonella serovars introduced onto the flowers of growing plants were recovered on and within the developing tomato fruit. Of all the Salmonella serovars tested, Montevideo appeared to be more adapted to survival within tomatoes and was recovered from 90% of the fruit screened. All of the Salmonella serovars could persist and grow when introduced onto unripened (green) tomato fruit. In general, growth (internal and external) was promoted at the high incubation temperature (25 degrees C) and high relative humidity (95%), although this was serovar dependent. The growth and persistence of Salmonella introduced on and into ripened (red) tomatoes was serovar dependent. Salmonella serovars Enteritidis, Typhimurium, and Dublin were less adapted to grow in or on intact red tomatoes than were serovars Hadar, Montevideo, or Newport. The results illustrated that a diverse range of Salmonella serovars can become established within and/or on preharvest tomatoes. The majority of Salmonella can grow and become established both on and within unripened tomatoes, but growth on ripened fruit was serovar dependent. The results provide a possible explanation why only a narrow range of Salmonella serovars are associated with foodborne illness outbreaks linked to tomatoes.

Untangling metabolic and communication networks: interactions of enterics with phytobacteria and their implications in produce safety

Aims:  To evaluate the efficacy of ultraviolet (UV) light (254 nm) combined with hydrogen peroxide (H2O2) to inactivate bacteria on and within fresh produce.

The tricks learnt by human enteric pathogens from phytopathogens to persist within the plant environment

Recent outbreaks of vegetable-borne gastrointestinal illnesses across the globe demonstrate that human enteric pathogens can contaminate produce at any stage of production. Interactions of enterics with native plant-associated microbiota influence the microbiological safety of produce by affecting the attachment, persistence and proliferation of human pathogens on plants. Supermarket surveys have revealed that bacteria, but not fungi or mechanical damage, promote the growth of Salmonella enterica on produce. Field and laboratory studies have indicated that some plant pathogenic bacteria and fungi facilitate the entry and internalization of human pathogens in plants. Conversely, some phytobacteria, including those involved in biocontrol of plant diseases, significantly inhibit attachment and plant colonization by non-typhoidal Salmonella and enterovirulent Escherichia coli by producing antibiotics or competing for nutrients in the phyllosphere. In this review, we attempt to elucidate the mechanisms of interactions between human enteric pathogens and plant-associated microbiota, and describe how these interactions affect produce safety.

Internalization of Human Pathogens within Growing Salad Vegetables

Through recent advances in our understanding of microbial:plant interactions it is becoming apparent that human pathogens, principally, Escherichia coli O157:H7 and Salmonella are adapted to survive in the plant environment. The aforementioned pathogens have surface epitopes that can bind to plant structures such as stomata to aid attachment. In addition, Salmonella is attracted and able to metabolize nutrients contained within the apoplastic fluid of plants. The question of internalization into the inner tissue of plants remains inconclusive largely because of the problems encountered in detecting low pathogen levels. Nevertheless, once internalized human pathogens can trigger and potentially evade plant defenses that are typically induced by phytopathogens. Although more research in this area is required, the hypothesis that human pathogens have adapted to the plant environment as part of their natural lifecycle appears to be supported.

A single DNA aptamer functions as a biosensor for ricin

’Department of Food Science, University of Guelph, Guelph, Ontario, Canada NIG 2W1, 2Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughbm.ough, Leicestershire, LEI2 5RD, UK, ’Division of Plant Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LEI2 5RD, UK and 4Division of Agricultural Sciences, School of Biosciences. University of Nottingham, Sutton Bonington Canzpus, Loughborough, Leicestershire, LEI2 5RD, UK

Inactivation of Escherichia coli O157:H7 and Salmonella on mung beans, alfalfa, and other seed types destined for sprout production by using an oxychloro-based sanitizer

The use of microorganisms or toxins as weapons of death and fear is not a novel concept; however, the modes by which these agents of bioterrorism are deployed are increasingly clever and insidious. One mechanism by which biothreats are readily disseminated is through a nations food supply. Ricin, a toxin derived from the castor bean plant, displays a strong thermostability and remains active at acidic and alkaline pHs. Therefore, the CDC has assigned ricin as a category B reagent since it may be easily amendable as a deliberate food biocontaminate. Current tools for ricin detection utilize enzymatic activity, immunointeractions and presence of castor bean DNA. Many of these tools are confounded by complex food matrices, display a limited dynamic range of detection and/or lack specificity. Aptamers, short RNA and single stranded DNA sequences, have increased affinity to their selected receptors, experience little cross-reactivity to other homologous compounds and are currently being sought after as biosensors for bacterial contaminants in food. This paper describes the selection and characterization of a single, dominant aptamer, designated as SSRA1, against the B-chain of ricin. SSRA1 displays one folding conformation that is stable across 4-63 °C (ΔG = -5.05). SSRA1 is able to concentrate at least 30 ng mL(-1) of ricin B chain from several liquid food matrices and outcompetes a currently available ELISA kit and ricin aptamer. Furthermore, we show detection of 25 ng mL(-1) of intact ricin A-B complex using SSRA1 combined with surface enhanced Raman scattering technique. Thus, SSRA1 would serve well as pre-analytical tool for processing of ricin from liquid foods to aid current diagnostics as well as a sensor for direct ricin detection.

Microbial population profiles of the microflora associated with pre‐ and postharvest tomatoes contaminated with Salmonella typhimurium or Salmonella montevideo

The efficacy of a stabilized oxychloro-based food grade sanitizer to decontaminate seeds destined for sprout production has been evaluated. By using mung bean seeds as a model system, it was demonstrated that the sanitizer could be used to inactivate a five-strain cocktail of Escherichia coli O157:H7 or Salmonella introduced onto beans at 10(3) to 10(4) CFU/g. Salmonella was more tolerant to stabilized oxychloro than was E. coli O157:H7, with sanitizer levels of >150 and >50 ppm, respectively, being required to ensure pathogen-free sprouts. The decontamination efficacy was also found to be dependent on treatment time (>8 h optimal) and the seed-to-sanitizer ratio (>1:4 optimal). Stabilized oxychloro treatment did not exhibit phytotoxic effects, as germination and sprout yields were not significantly (P > 0.05) different as compared with untreated controls. Although human pathogens could be effectively eliminated from mung beans, the aerobic plate count of native microflora on sprouts grown from treated seed was not significantly (P > 0.05) different from the controls. The diversity of microbial populations (determined through 16S rRNA denaturing gradient gel electrophoresis analysis) associated with bean sprouts was not significantly affected by the sanitizer treatment. However, it was noted that Klebsiella and Herbasprillum (both common plant endophytes) were absent in sprouts derived from decontaminated seed but were present in control sprouts. When a further range of seed types was evaluated, it was found that alfalfa, cress, flax, and soybean could be decontaminated with the stabilized oxychloro sanitizer. However, the decontamination efficacy with other seed types was less consistent. It appears that the rate of seed germination and putative activity of sanitizer sequestering system(s), in addition to other factors, may limit the efficacy of the decontamination method.